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\begin{document}

\title{The direct and legacy effects of drying-rewetting cycles on active and
relatively resistant soil carbon decomposition}



\author[1]{Shuai Zhang}%
\author[2]{Junjie Lin}%
\author[3]{Peng Wang}%
\author[4]{Biao Zhu}%
\affil[1]{Zhejiang University}%
\affil[2]{Chongqing Three Gorges University}%
\affil[3]{c.	Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology}%
\affil[4]{Peking University}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Global climate change is expected to increase the frequency of drought
and heavy precipitation, which could create more frequent
drying-rewetting cycles (DWC) in the soils. Although DWC effects on SOC
decomposition has been widely studied, the effect of DWC and the
subsequent legacy effect on the decomposition of different SOC pools is
still unclear. We conducted a 128-d laboratory incubation to investigate
the DWC effects by using soils from old-field for 15 years (OF,
representing active SOC), bare-fallow for 15 years (BF), and bare-fallow
for 23 years plus extra 815-d incubation (BF+, representing relatively
resistant SOC). The experiment included nine 10-d DWC of three
treatments: 1) constant-moisture at 60\% WHC, 2) mild DWC with 10-d
drying to 40\% WHC and rewetting to 80\% WHC, and 3) strong DWC with
10-d drying to 20\% WHC and rewetting to 100\% WHC. Following DWC
period, there was a 10-d stabilization period (adjusting all treatments
to 60\% WHC), and then a 28-d extended incubation. During DWC period,
the strong DWC had strong effect on CO2 release compared with the
constant-moisture control, reducing the SOC decomposition from OF by 8\%
and BF by 10\%, while increasing the SOC decomposition of BF+ by 16\%.
During extended period, both mild and strong DWC significantly increased
SOC mineralization of OF, but decreased that of BF and BF+. This legacy
effect compensated the changes in CO2 release during DWC period,
resulting in the minor response of SOC decomposition of OF and BF+ to
the DWC during the entire incubation.%
\end{abstract}%



\sloppy


\textbf{The direct and legacy effects of drying-rewetting cycles on
active and relatively resistant soil carbon decomposition}

Shuai Zhang\textsuperscript{a,b}, Junjie Lin\textsuperscript{a*}, Peng
Wang\textsuperscript{c}, Biao Zhu\textsuperscript{d*}

\begin{enumerate}
\tightlist
\item
  Chongqing Key Laboratory of Water Environment Evolution and Pollution
  Control in Three Gorges Reservoir, Chongqing Three Gorges University,
  Chongqing 404100, China
\item
  College of environmental and resource sciences, Zhejiang University,
  Hangzhou, Zhejiang 310058, China
\item
  Key Laboratory of Forest Ecology and Management, Institute of Applied
  Ecology, Chinese Academy of Sciences, Shenyang 110016, China
\item
  Institute of Ecology, College of Urban and Environmental Sciences, and
  Key Laboratory for Earth Surface Processes of the Ministry of
  Education, Peking University, Beijing 100871, China
\end{enumerate}

*Corresponding authors: Dr. Junjie Lin and Dr. Biao Zhu

E-mail addresses:\emph{junjielin@sanxiau.edu.cn}(J. Lin) and
\emph{biaozhu@pku.edu.cn}(B. Zhu)

\textbf{Abstract:} Global climate change is expected to increase the
frequency of drought and heavy precipitation, which could create more
frequent drying-rewetting cycles (DWC) in the soils. Although the DWC
effects on soil organic carbon (SOC) decomposition has been widely
studied, the effect of DWC and the subsequent legacy effect on the
decomposition of different SOC pools is still unclear. We conducted a
128-d laboratory incubation to investigate the DWC effects by using
soils from old-field for 15 years (OF, representing active SOC),
bare-fallow for 15 years (BF), and bare-fallow for 23 years plus extra
815-d incubation (BF+, representing relatively resistant SOC). The
experiment included nine 10-d DWC of three treatments: 1) one mean
constant-moisture at 60\% water-holding capacity (WHC), 2) a mild DWC
with 10-d drying to 40\% WHC and immediately rewetting to 80\% WHC, and
3) a strong DWC with 10-d drying to 20\% WHC and immediately rewetting
to 100\% WHC. Following the DWC period (0-90 d), there was a 10-d
stabilization period (adjusting all treatments to 60\% WHC), and then a
28-d extended incubation under the constant moisture of 60\% WHC. During
the DWC period, the strong DWC had a strong effect on
CO\textsubscript{2} release compared with the constant-moisture control,
reducing the SOC decomposition from OF by 8\% and BF by 10\%, while
increasing the SOC decomposition of BF+ by 16\%. In addition, during the
extended period, both mild and strong DWC significantly increased SOC
mineralization of OF, but decreased that of BF and BF+. This legacy
effect induced by DWC compensated the changes in CO\textsubscript{2}
release during the DWC period, resulting in the minor response of SOC
decomposition of OF and BF+ to the DWC during the entire incubation.
Together, DWC could create both direct and legacy effects, and these
effects vary with DWC intensity and SOC pools.

\textbf{Keywords} : Drying-rewetting; Soil carbon pool; Soil organic
matter; Soil respiration; Legacy effect

\textbf{Abbreviations} : DWC -- drying-rewetting cycles; OF --
old-field; BF -- bare-fallow; BF+ -- bare-fallow + incubation; C --
carbon; CO\textsubscript{2} -- carbon dioxide; SOC -- soil organic
carbon; MBC -- microbial biomass carbon; DOC -- dissolved organic
carbon; TC -- total carbon; TN -- total nitrogen; C/N -- the ratio of
carbon to nitrogen; F:B -- the ratio of fungi to bacteria.

\textbf{1. Introduction}

Global climate models predict increases in atmospheric carbon dioxide
(CO\textsubscript{2}) concentration and surface temperature (IPCC,
2013), which are likely to alter regimes of global hydrologic cycling,
including an increase in the number and severity of droughts and heavier
precipitation between drought periods (Dai, 2013; Donat et al., 2016).
Soil moisture is a key factor controlling the microbial decomposition of
soil organic carbon (SOC) (Moyano et al., 2013). When the soil is dry,
microbial activity and substrate diffusion will be restricted, and soil
respiration will be reduced (Schimel, 2018). The rapid precipitation
after drought usually increases mineralization of SOC, creating a large
pulse of CO\textsubscript{2}(Birch, 1958; Kim, et al., 2012). The
CO\textsubscript{2} pulse has been attributed to the rapid consumption
of microbial necromass as available substrates and released microbial
osmoregulatory substances in response to water stress (Blazewicz et al.,
2014; Chowdhury et al., 2019; Warren, 2016). In addition, the sudden
influx of water at rewetting can expose previously inaccessible carbon
(C) to microorganisms through aggregates breakdown (Denef et al., 2001;
Najera et al., 2020; Schimel et al., 2011). This rewetting-driven
CO\textsubscript{2} pulse can be sustained for more than 20 days
(Canarini et al., 2017) and elevated by as much as 475\% relative to a
constant-moisture soil (Fierer and Schimel, 2003). Therefore, soil
drying-rewetting cycles (DWC) are considered an essential environmental
factor regulating C cycle in terrestrial ecosystems (Borken and Matzner,
2009; Fierer and Schimel, 2002; Muhr et al., 2008; Schimel, 2018; Zhu
and Cheng, 2013).

Although a large number of studies have examined the effects of DWC on
soil C decomposition, DWC increased (Butterly et al., 2009; Miller et
al., 2005; Shi and Marschner, 2014; Xiang et al., 2008; Yemadje et al.,
2017), decreased (Shi and Marschner, 2014), or did not change (Yemadje
et al., 2017) cumulative C decomposition relative to constant-moisture
control in previous studies. Zhang et al. (2020) indicated in a recent
meta-analysis that relative to a constant-moisture control (with the
same mean value), the rewetting-driven CO\textsubscript{2} pulse can
fully compensate the reduced CO\textsubscript{2} emission during the
drying phase, thus DWC did not change cumulative C decomposition. They
also commented that the changes in cumulative C loss depend on DWC
intensity. Intensified drought usually triggers a stronger
CO\textsubscript{2} pulse after rewetting (Barnard et al., 2015; Li et
al., 2018), because a severer drought may lead to a higher frequency of
microbial death and accumulate more compatible solutes, which can
contribute largely to the CO\textsubscript{2} pulse (Barnard et al.,
2020; Guo et al., 2012). Moreover, there may be a drought threshold,
beyond which the inaccessible C will be accessed, thereby contributing
to the subsequent CO\textsubscript{2} pulse (Canarini et al., 2017;
Homyak et al., 2018). Moreover, DWC can promote the release of old C
that had been occluded in soils for more than 600 years (Schimel et al.,
2011). However, due to the difficulties in separation of decomposition
of SOC pools with different turnover times, few studies have evaluated
the vulnerability of different SOC pools to DWC. Therefore, due to the
large proportion of stable C in the total soil C pool, clarifying its
response to climate change is essential for predicting future changes in
the global C cycle.

Soil organic carbon can be divided into three pools with different
physiochemical characteristics and turnover times (Davidson and
Janssens, 2006; Lin et al., 2015). The most active fraction is annually
cycling, active SOC, which mainly consists of microbial biomass and
plant detritus, and has a fast turnover time from weeks to years,
contributing to approximately 0\textasciitilde{}5\% of the total SOC
pool (Parton et al., 1987; Semenov et al., 2018). It hardly contributes
much to the global C cycle due to its small size and very fast turnover
time. The most inert fraction is millennially cycling, passive (inert)
SOC, which is mainly composed of black carbon or humic substances and
has a turnover time even more than centuries, accounting for about
10\textasciitilde{}40\% of the total SOC pool (Schmidt et al., 2011).
Furthermore, the more critical pool is the decadally cycling, relatively
resistant (intermediate, slow) SOC, which is the dominant component of
the total SOC pool, accounting for around 60\textasciitilde{}80\% of the
total SOC pool, and has a turnover time of decades. It is generally
believed that different microbial communities use different soil
substrates (Berg and McClaugherty, 2008; Xu et al., 2015), and bacteria
are considered more responsible for consuming readily available
substrates (Moore-Kucera and Dick, 2008), while fungi are believed to
have the ability to decompose more stable SOC (Xu et al., 2015). Fungi
display an overall higher resistance to drying and subsequent rewetting
processes than bacteria (Barnard et al., 2015; Yuste et al., 2011). As a
consequence, relatively resistant SOC may be more susceptible to DWC due
to the higher resistance of fungi to drought and thus the smaller
reduction in C decomposition during the drying process (de Vries et al.,
2018; Zhang et al., 2020).

Furthermore, previous conditions can affect current processes, which is
called the legacy effect (Monger et al., 2015). The DWC-driven legacy
effect can compensate for 14\% of the decrease of cumulative C
decomposition during the DWC period (Li et al., 2018). However, the
legacy effect of DWC on the decomposition of different SOC pools remains
unclear. Thus, the direct and legacy effects of DWC on SOC pools with
different turnover times remain uncertain and require further
investigation.

To fill this knowledge gap, we use soils from three plots in a long-term
experimental field to explore the direct and legacy effects of DWC on
active and relatively resistant soil C decomposition. In our study, the
direct effect represents the effect of DWC on C decomposition in the DWC
period, and the legacy effect denotes the effect of previous DWC on C
decomposition in the post-DWC incubation period with constant moisture.
We conducted a laboratory incubation experiment of 128 days, combining
three water regimes, nine 10-d DWC (in a row) and one 28-d extended
period, to investigate the direct and legacy effects of DWC on active
and relatively resistant soil C decomposition. We hypothesized that (i)
compared to the constant-moisture control, the DWC with intensified
drought would stimulate more C emission during the entire
drying-rewetting period due to the strong CO\textsubscript{2} pulse
after rewetting; (ii) the relatively resistant SOC (the bare-fallow and
the bare-fallow+incubation soils) would be more vulnerable to DWC than
the active SOC (the old-field soil) due to the higher resistance of
fungi in the relatively resistant SOC to DWC; and (iii) the DWC could
create a legacy effect on soil C decomposition due to the changes in
microbial biomass and labile substrates.

\textbf{2.} \textbf{Materials and methods}

\textbf{2.1. Site description and soil sampling}

Soils were sampled from a long-term experimental field located in the
Shenyang station of Chinese Academy of Sciences, Liaoning province,
Northeastern China (41\selectlanguage{ngerman}°32\selectlanguage{english}' N, 122deg23\selectlanguage{english}' E). The climate is typical
continental monsoon, with a mean annual temperature of 7.9degC and mean
annual precipitation of 604 mm
(1981-2018,\emph{https://power.larc.nasa.gov}). The long-term old-field
(OF) and bare-fallow (BF) treatments were established in 2003 and were
paddy fields before treatments. The OF plot received fresh organic input
for 15 years (2003-2018) with freely growing weeds but no anthropogenic
interference. The BF plot was kept free of organic input for 15 years
(2003-2018) by frequent hand weeding. Soils from these OF and BF
treatments were sampled in 2018. Another BF plot had been kept free of
organic input for 23 years until soil sampling for this study (Zhang et
al., 2021). Soils were collected from the plow layer (0-20 cm depth) in
the long-term experimental field. Within each plot, soils were
sub-sampled at five randomly selected sites and then homogenized into
one sample. Prior to the incubation experiment, soil samples were
air-dried, sieved to 2 mm, and homogenized thoroughly. Visible roots,
plant detritus, and stones were removed carefully by handpicking.

\textbf{2.2. Experimental design and soil incubation}

The soil from the OF treatment (15 year-old) represents active SOC. The
soils from the BF treatment for 15 years and BF treatment for 23 years
plus an extra 815 days of laboratory incubation (BF+, the additional
incubation is to further consume active organic carbon) represent
relatively resistant SOC (Zhang et al., 2017). Therefore, we refer to
the OF SOC as active C, the BF and the BF+ SOC as relatively resistant C
in this study. Although all three soils may still contain various
components with different turnover times and protection mechanisms, here
we operationally used the three soils to represent active and relatively
resistant SOC (Conant et al., 2008; Townsend et al., 1997), and compared
the relative vulnerability of these SOC pools to the direct and legacy
effects of drying-wetting cycles for two reasons. First, we measured the
proportion of active C (dissolved organic carbon/total carbon, DOC/TC)
and found that the proportion of active C in the OF soil is the highest,
and that in the BF+ soil is the lowest (Table 1). Second, we measured
the chemical composition of SOC of these three soils and found that the
molecular index of SOC stability (or resistance) in the OF soil is the
lowest, and that in the BF+ soil is the highest (Fig. 2). In addition,
Barre et al. (2010) constrained a three-pool-model using observed soil
organic C data in long-term BF soils and found that most of the organic
C are stable C (turnover time of several centuries or more). Basic
properties of these three soils can be found in Table 1 and Zhang et al.
(2017).

The incubation experiment consisted of a nine 10-d DWC period and a 28-d
extended period, with a 10-d stabilization period between them (Fig. 1).
In the DWC period (0-90 d), soils were subjected to three water regimes,
including one constant-moisture control (mean moisture content: 60\%
WHC), one mild DWC treatment (80-40\% WHC), and one strong DWC treatment
(100-20\% WHC), each with three replicates. After the 90-d DWC period,
all treatments were adjusted to constant moisture of 60\% WHC for ten
days to a consistent state (90-100 d). All soils were kept at 60\% WHC
during the subsequent 28-d extended period (100-128 d).

For each soil, a subsample of 60 g (dry weight basis) was placed in a
1-L incubation jar at 60\% WHC with deionized water, and the soils were
pre-incubated at 22degC for 10 days in the dark. To achieve soil drying,
45 g and 20 g silica gel were placed in the incubation jars for 100-20\%
WHC and 80-40\% WHC treatments, respectively (Fig. S1). These two
weights were chosen because our pre-experimental data showed that these
two weights of silica gel could accelerate the loss of soil moisture to
the expected levels (i.e. 20\% and 40\% WHC) in 10 days. Inside each
jar, 10 ml 0.5 M NaOH solution was used to trap CO\textsubscript{2} from
SOC decomposition. At the end of each drying period, the NaOH solution
and silica gel were replaced, and the soil samples were rapidly rewetted
by adding deionized water with a syringe. The trapped
CO\textsubscript{2}-C in the NaOH solutions were immediately measured in
the form of total inorganic C using a Lotix combustion TOC analyzer
(Teledyne Tekmar, USA), and the silica gel was dried in an oven at 105degC
overnight to regenerate it. After replacing the silica gel and NaOH
solution, the jars were sealed immediately with gas-tight lids and kept
at 22degC in the dark. A previous study proved that silica gel does not
absorb CO\textsubscript{2}(Harrison-Kirk et al., 2013). Three blanks
without soil were incubated at the same condition to quantify the amount
of C in the air and initial NaOH solution. Prior to measuring the total
inorganic C, the NaOH solutions in the DWC treatments were adjusted back
to 10 ml with deionized water.

After the 90-d DWC period, all soils were adjusted to a constant
moisture content of 60\% WHC and maintained for 10 days (stabilization
period, 90-100 d). Note that we did not measure respiration for the
stabilization period. On day 100, we did a destructive soil sampling.
Specifically, 8 g subsamples were used to determine dissolved organic
carbon (DOC) and microbial biomass carbon (MBC), 10 g subsamples were
stored at -80degC until the measurement of microbial biomass, and 10 g
subsamples were incubated for another 28 d (extended period, 100-128 d)
in which the CO\textsubscript{2}-C in the NaOH solutions were measured
on day 103, 107, 114, 121 and 128.

In total, we incubated 30 jars ((3 x 3 + 1) x 3) consisting of three
soils (OF, BF and BF+ soils), two DWC treatments (100-20\% and 80-40\%
WHC) and one constant-moisture control treatment (60\% WHC), one blank,
and three replicates for each treatment.

\textbf{2.3. Soil property analyses}

Soil total C and N (nitrogen) contents were measured using an elemental
analyzer (EA 3000, Euro Vector, Milan, Italy). The pH was measured in a
1:2.5 soil-water ratio with a pH meter (PHS-3C, LEICI, China). The
particle size distribution was examined using the hydrometer method
(Bouyoucos, 1962). The WHC was measured by placing a
100-cm\textsuperscript{3}cutting ring filled with thoroughly wetted soil
for 24 h on another cutting ring filled with corresponding air-dried
soil and allowed to drain for 8 h.

Soil MBC content was measured using the chloroform fumigation extraction
method (Vance et al., 1987). Briefly, a soil sample was divided into two
sets (4 g per set), one set was fumigated with chloroform for 24 h in
the dark, and another set was unfumigated. Then the fumigated and
unfumigated samples were extracted with 20 ml 0.5 M
K\textsubscript{2}SO\textsubscript{4} in a 1:5 ratio. The extracts were
filtered through 0.45 \selectlanguage{greek}μ\selectlanguage{english}m syringe filter (Siyan Biotechnology, China) and
were determined in the form of total organic C using a Lotix combustion
TOC analyzer (Teledyne Tekmar, Mason, OH, USA). Soil MBC was calculated
from the K\textsubscript{2}SO\textsubscript{4}-extractable organic C
between the fumigated and unfumigated samples with a conversion factor
of 0.45. Total organic C in unfumigated samples represents DOC.

\textbf{2.4. Fourier-transform infrared spectroscopy analysis}

The molecular composition of SOC was characterized by Fourier transform
infrared (FTIR) spectroscopy (Thermo Fisher Scientific Nicolet iS10
spectrometer, USA) according to Demyan et al. (2012). Briefly, 2 mg
air-dried ground soil samples (\textless{}0.147 mm) were pressed into
100 mg potassium bromide (KBr) pellets (64 scans within the spectral
range 4000-400 cm\textsuperscript{-1} at a resolution of 4
cm\textsuperscript{-1} were averaged). Band maxima at wavenumbers around
2930, 2850, 1635, 1430 and 1040 cm\textsuperscript{-1} were selected and
were assigned to a range of organic functional groups (with limited
mineral interference). The peak areas of these five bands were
integrated using a tangential baseline from the onset to the offset of
each peak, and their relative peak areas of each band were calculated to
evaluate the assignments of soil C functional groups (Table S1). The
ratio of the peak areas at 1635 and 2930
cm\textsuperscript{-1}(rA1635/rA2930 ratio) was defined as the
resistance index and often taken as an indicator of SOC stability
(Demyan et al., 2012; Ernakovich et al., 2015; Hou et al., 2019).

\textbf{2.5. Polymerase chain reaction (PCR) amplification}

The copy numbers of fungi and bacteria were measured by PCR
amplification following Xia et al. (2011). DNA was extracted from 0.5 g
soil using the NucleoSpin Soil DNA extraction kit (Macherey-Nagel,
Duren, Germany). DNA extracts were stored at -20. The 16s rRNA gene, a
molecular marker for bacteria, was amplified using 515F/907R (forward
5'-GTGCCAGCMGCCGCGG-3'; reverse 5'-CCGTCAATTCMTTTRAGT-3'). PCRs were
performed in 20 \selectlanguage{greek}μ\selectlanguage{english}l containing 1 \selectlanguage{greek}μ\selectlanguage{english}l of TB Green Fast qPCR Mix, 2.5 \selectlanguage{greek}μ\selectlanguage{english}l of
each primer (10 \selectlanguage{greek}μ\selectlanguage{english}M), 1 \selectlanguage{greek}μ\selectlanguage{english}l of DNA template and 20 \selectlanguage{greek}μ\selectlanguage{english}l of molecular biology
quality water. The amplifications were performed in a CFX-96 (Bio-Rad)
following the thermal program: (1) 95\selectlanguage{ngerman}°C for 5 min; (2) 40 cycles at 95°C
for 45 s, 51°C for 45 s and 72°C for 60 s; and (3) 72°C for 5 min.

The ITS gene, a molecular marker for fungi, was amplified using
ITS1F/ITS2R (forward 5'-CTTGGTCATTTAGAGGAAGTAA-3'; reverse
5'-GCTGCGTTCTTCATCGATGC-3'). PCRs were performed in 20 \selectlanguage{greek}μ\selectlanguage{english}l containing 1
\selectlanguage{greek}μ\selectlanguage{english}l of TB Green Fast qPCR Mix, 2.5 \selectlanguage{greek}μ\selectlanguage{english}l of each primer (10 \selectlanguage{greek}μ\selectlanguage{english}M), 1 \selectlanguage{greek}μ\selectlanguage{english}l of DNA
template and 20 \selectlanguage{greek}μ\selectlanguage{english}l of molecular biology quality water. The
amplifications were performed in a CFX-96 (Bio-Rad) following the
thermal program: (1) 95\selectlanguage{ngerman}°C for 5 min; (2) 40 cycles at 95°C for 45 s,
55°C for 45 s and 72°C for 60 s; and (3) 72°C for 5 min. The F:B values
were calculated using the ratio of the fungal-to-bacterial gene copy
numbers. qPCR cannot provide an estimation of the F:B biomass ratio
because different taxa contain an unknown number of copies of the rDNA
operon in their genomes. But it provides information about the
differences in the relative abundance of fungi and bacteria across soil
samples (Fierer et al., 2005).

\textbf{2.6. Statistical analysis}

For all response variables, significant differences (\emph{P}\textless{}
0.05) among water regimes in each soil and significant differences among
soils in each water regime were assessed by one-way analysis of variance
(ANOVA). The effects of water regimes (one constant-moisture control
treatment at 60\% WHC and two DWC treatments of 100-20\% and 80-40\%
WHC), soil treatments (OF, BF and BF+) and their interactions on C
decomposition, fungal-to-bacterial ratio (F:B), MBC and DOC were
assessed by linear mixed models (LMM). For the LMM, soil treatment and
water regime were considered as fixed factors, and sample identity was
regarded as a random factor. All statistical analyses were performed
using IBM SPSS Statistics 20 (IBM SPSS Inc.), and graphs were drawn
using SigmaPlot 14.0 (Systat Software Inc.).

\textbf{3. Results}

\textbf{3.1. Effects of long-term field treatments on soil properties}

The 15-year BF and 23-year BF+ treatments significantly decreased soil C
and N contents, but did not significantly affect the C/N ratio compared
with the OF treatment (\emph{P} \textless{} 0.05, Table 1). Soil clay
content was lower in the BF and BF+ treatments in comparison with the OF
treatment, and soil silt content was higher in the BF+ treatment than
that of the OF treatment. Furthermore, the BF and BF+ treatments
significantly decreased soil DOC content, and the BF+ treatment
significantly reduced DOC/TC ratio compared with the OF treatment. It
implies lower active C (DOC) content in the BF and BF+ soils due to the
long-term removal of plant inputs. Although soil pH of the BF and BF+
treatments and soil WHC of the BF+ treatment were significantly lower
than those of the OF treatment, the differences among soil pH did not
exceed 0.3 units and the differences among soil WHC did not exceed 1\%.

\textbf{3.2. Molecular index of SOC stability}

Fourier--transform infrared (FTIR) spectroscopy of three soils generally
featured common peaks but showed different intensities (Fig. S2). The
rA1635 of BF+ soil was significantly lower than that of OF soil, and the
order of the rA2930 of the three soils was OF \textgreater{} BF
\textgreater{} BF+ (\emph{P}\textless{} 0.05, Fig. 2a, b and Table S1).
The order of the resistance index (ratio of rA1635/rA2930) of the three
soils was BF+ \textgreater{} BF \textgreater{} OF (\emph{P} \textless{}
0.05, Fig. 2c). The trend of resistance index (rA1635/rA2930) indicated
that the molecular index of SOC stability (or resistance) was highest in
BF+ soil, followed by BF soil, and finally OF soil.

\textbf{3.3. Dynamics of soil carbon decomposition}

During the DWC period, soil C mineralization rates declined rapidly
after the first cycle (0-10 d) for all water regimes, and then decreased
slowly with the increasing number of DWC (Fig. 3a-c). For OF soils,
cumulative soil C mineralization in 60\%, 80-40\%, and 100-20\% WHC
treatments were 28.0, 27.1, and 25.7 mg CO\textsubscript{2}-C
g\textsuperscript{-1} SOC, respectively. For BF soils, cumulative soil C
mineralization in 60\%, 80-40\%, and 100-20\% WHC treatments were 24.0,
22.3, and 21.6 mg CO\textsubscript{2}-C g\textsuperscript{-1} SOC,
respectively. For BF+ soils, cumulative soil C mineralization in 60\%,
80-40\%, and 100-20\% WHC treatments were 10.8, 11.9, and 12.5 mg
CO\textsubscript{2}-C g\textsuperscript{-1} SOC, respectively (Fig.
3d-f, Fig. 4a). Compared to the constant-moisture control treatment
(60\% WHC), the 100-20\% WHC treatment decreased cumulative C
mineralization of OF and BF soils by 8\% and 10\%, respectively, but
increased that of BF+ soil by 16\%. The 80-40\% WHC treatment did not
significantly change cumulative C mineralization of all three soils
relative to the constant-moisture control treatment (Fig. 4a and Table
S2). There was also an interactive effect of water regime and soil
treatment on cumulative C mineralization during the DWC period (\emph{P}
=0.001, Table S3).

During the extended period, cumulative soil C mineralization of 60\%,
80-40\%, and 100-20\% WHC treatments in OF soil were 5.0, 5.9, and 5.8
mg CO\textsubscript{2}-C g\textsuperscript{-1} SOC, respectively. For BF
soil, cumulative soil C mineralization in 60\%, 80-40\%, and 100-20\%
WHC treatments were 5.8, 3.8, and 4.0 mg CO\textsubscript{2}-C
g\textsuperscript{-1} SOC, respectively (Fig. 4b and Table S2). For BF+
soil, cumulative soil C mineralization in 60\%, 80-40\%, and 100-20\%
WHC treatments were 4.9, 2.0, and 1.3 mg CO\textsubscript{2}-C
g\textsuperscript{-1} SOC, respectively (Fig. 4b and Table S2). Compared
to the constant-moisture control treatment, both DWC treatments (80-40\%
and 100-20\% WHC) increased the cumulative C mineralization in OF soil
but decreased it in BF and BF+ soils. Specifically, the 80-40\% WHC
treatment increased C mineralization by 18\% in OF soil and decreased it
by 35\% and 59\% in BF and BF+ soils, respectively; The 100-20\% WHC
treatment increased C mineralization by 16\% in OF soil and decreased it
by 31\% and 74\% in BF and BF+ soils, respectively (Fig. 4b and Table
S2). There was also an interactive effect of water regime and soil
treatment on cumulative C mineralization during the extended period
(\emph{P} =0.001, Table S3).

During the entire incubation period (0-90 d and 100-128 d), the 80-40\%
and 100-20\% WHC treatments decreased cumulative C mineralization of BF
soil by 12\% and 14\%, respectively, but did not significantly change
cumulative C mineralization of OF and BF+ soils compared with the
constant-moisture control treatment (Fig. 4c and Table S2).

\textbf{3.4. Soil MBC, DOC, and F:B ratio}

Drying-rewetting cycles had significant impacts on MBC (Fig. 5a). The
80-40\% and 100-20\% WHC treatments decreased the MBC content of BF and
BF+ soils. The 100-20\% WHC treatment increased the MBC content of OF
soil (Fig. 5a). Soil DOC content did not differ among water regimes in
all three soils (Fig. 5b).

Both 80-40\% and 100-20\% WHC treatments increased F:B ratio in the BF+
soil (Fig. 5c). For constant moisture treatment at 60\% WHC, F:B ratio
was lowest in the OF soil. Both MBC and DOC had a positive linear
relationship with cumulative C mineralization during the extended period
across the three soils (Fig. 6).

\textbf{4. Discussion}

Our results indicated that DWC had contrasting effects on the
decomposition of different SOC pools. The strong DWC (100-20\% WHC)
increased C release from the BF+ soil, but decreased C release from the
OF and BF soils, respectively. The DWC-driven legacy effect on C release
can last for at least 38 d. Both the amount of microbial biomass and
labile substrates contributed to this legacy effect. When considering
both the extended and the DWC periods, the 80-40\% and 100-20\% WHC
treatments had no significant effect on SOC decomposition of OF and BF+
soils, but decreased SOC decomposition of BF soil by 12\% and 14\%,
respectively, compared with the constant-moisture control treatment.
Taken together, these results demonstrate the importance of DWC in
modulating SOC dynamics and show the different vulnerability of
different SOC pools to DWC.

\textbf{4.1. Direct effects of DWC on soil carbon mineralization}

As we hypothesized, the strong DWC with intensified drought had a
stronger impact on C emission, showing that compared to the
constant-moisture control treatment (60\% WHC), the 100-20\% WHC
treatment decreased cumulative C mineralization during the DWC period in
OF and BF soils and increased it in BF+ soil, while the 80-40\% WHC
treatment did not significantly change the C emission of all three soils
(Fig. 4a). This is because the 80-40\% WHC treatment might not cause as
much osmotic shock and cell lyses as the strong DWC treatment (100-20\%
WHC in this study) during the drying period (Guo et al., 2012). Also,
the 80-40\% WHC treatment might not reach the drought threshold, and the
accessible C from physical and physiological mechanisms after the mild
drought (to 40\% WHC) may not contribute much to the subsequent
CO\textsubscript{2} pulse (Barnard et al., 2020; Slessarev et al.,
2020).

Although the 100-20\% WHC treatment decreased cumulative C
mineralization during the entire DWC period (0-90 d) in OF and BF soils,
this reduction in C emission of the 100-20\% WHC treatment only occurred
in two cycles and did not significantly affect C mineralization in other
cycles (Table S2). This result was consistent with a meta-analysis of
Zhang et al. (2020), which showed that the rewetting-driven
CO\textsubscript{2} pulse can fully compensate the reduced
CO\textsubscript{2} emission during the drying period.

For BF+ soil, the 100-20\% WHC treatment increased cumulative C
mineralization by 16\% relative to the mean constant-moisture control
treatment (Table S2). Since few active SOC in the BF+ soil was left
after 25-year BF treatment and 815-d laboratory incubation, the
microbial community in the BF+ soil is more dominated by fungi (Fig.
5c). Fungi are considered more resistant to drought and osmotic stress
than bacteria due to their multicellular hyphal networks allowing remote
access to water in the soil (Barnard et al., 2013). Therefore, the
reduction in C mineralization of the soil with more fungi (BF+ soil)
during the drying period may be smaller, and the cumulative C
mineralization may be higher during the whole DWC period compared to the
soil dominated by bacteria (OF soil). Moreover, DWC can result in a
microbial composition shift toward more desiccation tolerant. For
example, sensitive microorganisms die or become inactive during drought
and are difficult to recover, thereby supplying substrates for more
drought-tolerant surviving microorganisms (Meisner et al., 2021).

\textbf{4.2. Legacy effects of DWC on soil carbon mineralization}

Both 80-40\% and 100-20\% WHC treatments increased C mineralization in
OF soil but decreased it in BF and BF+ soils compared to the mean
constant-moisture control treatment during the extended period (Fig.
4b). This result indicated that the repeated DWC could cause a legacy
effect on soil C mineralization, and this legacy effect can last for at
least 38 d (including 10-d stabilization period and 28-d extended
period). Butterly et al. (2011) implied that this legacy effect can last
for more than 35 d, and Li et al. (2018) believed that after 4 times of
DWC, this legacy effect can last for at least 60 d.

Compared to the mean constant-moisture treatment (60\% WHC), the MBC
content at the start of the extended period (100 d) was higher in the
100-20\% WHC treatment for OF soil (Fig. 5a), while was lower in both
80-40\% and 100-20\% WHC treatments for BF and BF+ soils (Fig. 5a).
Moreover, there was a positive correlation between MBC and cumulative C
mineralization during the extended period across the three soils (Fig.
6a). These results suggested that the changes in microbial biomass
affected the legacy effect of DWC on soil C mineralization.
Additionally, DOC was also positively correlated with cumulative C
mineralization during the extended period across the three soils (Fig.
6b), indicating that the amount of microbial biomass and labile
substrates (i.e. DOC) both contributed to the legacy effect of DWC on
soil C mineralization. Notably, the fact that both MBC and DOC were
positively correlated with cumulative C mineralization during the
extended period is likely due to the positive linear relationship
between MBC and DOC (Fig. S4). Moreover, the DWC-driven changes of soil
microbial community composition may also contribute to this legacy
during the extended period (Meisner et al., 2021). Together, these
results are consistent with the finding of Li et al. (2018) who showed
that the legacy effect during the extended period is attributed to the
amount of plant litter (as substrate) and microbial biomass.

Taking together, both mild and strong DWC treatments had a minor effect
on SOC decomposition in OF and BF+ soils, respectively, but decreased
SOC decomposition in BF soil by 12\% and 14\%, respectively (Fig. 4c).
However, it should be noted that these results need to be interpreted
with caution, because we did not measure the C release during the
stabilization period (90-100 d) and the extended period (100-128 d, 28
days) may not be long enough. Future studies can use a longer extended
period and measure microbial community composition for a more
mechanistic understanding of the legacy effect of DWC on soil C release.

\textbf{5. Conclusion}

This study explored the direct and legacy effects of DWC on active and
relatively resistant SOC decomposition by using three soils from a
long-term experimental field as well as an incubation experiment.
Compared to the mean constant-moisture treatment (60\% WHC), the strong
DWC treatment (100-20\% WHC) had minor effects on SOC mineralization of
OF soil and BF soil in most of the cycles, but stimulated SOC
mineralization of BF+ soil. This result suggests that the relatively
resistant SOC is more vulnerable to DWC than active SOC during the DWC
period (0-90 d). Moreover, the DWC had a legacy effect on SOC
decomposition during the extended period (100-128 d), being positive for
active SOC but negative for relatively resistant SOC. Both MBC and DOC
may contribute to this legacy effect. However, when both direct effect
and legacy effect were considered, the strong DWC did not significantly
affect the cumulative SOC decomposition of OF and BF+ soils, but had a
negative effect on that of BF soil during the entire incubation period.
Taken together, our study demonstrated that DWC could create both direct
and legacy effects on SOC decomposition, and these effects vary with DWC
intensity and SOC pools. These findings suggest that more frequent
alternating drought and rapid precipitation in the future may have a
minor or even negative effect on soil C loss.

\textbf{Acknowledgements}

This work was supported by the National Natural Science Foundation of
China (31770529 and 31988102), the Science and Technology Research
Program of Chongqing Municipal Education Commission (KJZD-K202001203,
KJZD-K202003501) and the Chongqing Science and Technology Commission
(cstc2020jcyj-msxmX0095, WEPKL2018ZD-05). We thank Dr. Weixin Cheng (UC
Santa Cruz) for suggesting the paired old-field and bare-fallow soils
and Dr. Xiuwei Zhang (Taizhou University) for providing the
bare-fallow+incubation soils used in this study. We thank Xi Chen,
Shuang Zhou, and Zhuolin Yu for laboratory assistance. We also thank Dr.
Bing Yang of Chengdu Institute of Biology, Chinese Academy of Sciences
for his help in statistical analysis.

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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\textbf{Table 1} Physical and chemical properties of the soils. Values
represent means of three replicates with standard deviations in
parenthesis. Means within each row followed by different letters
represent a significant difference (\emph{P} \textless{} 0.05). SOC,
soil organic carbon; WHC, water-holding capacity; DOC, dissolved organic
carbon. OF, old-field; BF, bare-fallow; BF+, bare-fallow+incubation.
These soils contain little inorganic carbon and soil total C is
dominated by SOC.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Parameters & OF & BF & BF+\tabularnewline
\midrule
\endhead
Total C (\%) & 2.25 (0.26) a & 1.30 (0.05) b & 1.05 (0.01)
b\tabularnewline
Total N (\%) & 0.17 (0.00) a & 0.11 (0.00) b & 0.08 (0.00)
c\tabularnewline
C/N & 13.6 (1.6) a & 12.3 (0.4) a & 13.3 (0.4) a\tabularnewline
pH\textsubscript{H2O} & 6.74 (0.01) a & 6.50 (0.03) b & 6.52 (0.02)
b\tabularnewline
Sand (\%) & 49.1 (3.3) a & 50.1 (1.5) a & 46.1 (1.1) a\tabularnewline
Silt (\%) & 31.8 (2.8) a & 36.0 (2.2) ab & 40.1 (1.1) b\tabularnewline
Clay (\%) & 19.1 (1.2) a & 13.9 (1.0) b & 13.9 (0.3) b\tabularnewline
WHC (\%) & 32.7 (0.3) a & 32.4 (0.1) ab & 31.7 (0.3) b\tabularnewline
DOC (mg C kg\textsuperscript{-1} soil) & 187.4 (5.5) a & 96.4 (5.7) b &
72.0 (2.7) c\tabularnewline
DOC/TC (\%) & 0.84 (0.11) a & 0.74 (0.04) ab & 0.69 (0.03)
b\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig/Fig}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fih/Fih}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fii/Fii}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fij/Fij}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fik/Fik}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fil/Fil}

\end{center}
\end{figure}

\selectlanguage{english}
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